1. Field of the Invention
This invention relates to compositions that are radiolabeled scintigraphic imaging agents, methods of using these compositions and methods for producing such radiolabeled compositions. Specifically, the invention relates to technetium-99m labeled scintigraphic imaging agents that are compositions of a polysulfated glycan or mixture thereof and a compound comprising a polybasic moiety covalently linked to a radiolabel binding moiety radiolabeled with technetium-99m (Tc-99m). Methods and kits for making such compositions, and methods for using such compositions to image sites of infection and inflammation in a mammalian body are also provided.
2. Description of the Prior Art
There is a clinical need to be able to determine the location and/or extent of sites of focal or localized infection and inflammation. In a substantial number of cases conventional methods of diagnosis (such as physical examination, x-ray, CT and ultrasonography) fail to identify such sites (e.g., an abscess). Although biopsy may be resorted to, it is preferable to avoid such invasive procedures, at least until they are diagnostically appropriate to identify the pathogen responsible for an abscess at a known location. Identifying the site of such "occult" infection is important because rapid localization and identification of the problem is critical to effective therapeutic intervention.
In the field of nuclear medicine, certain pathological conditions can be localized or the extent of such conditions determined by imaging the internal distribution of administered radioactively-labeled tracer compounds (i.e. radiotracers or radiopharmaceuticals) that accumulate specifically at the pathological site. A variety of radionuclides are known to be useful for radioimaging, including .sup.67 Ga, .sup.99m Tc (Tc-99m), .sup.111 In, .sup.123 I, .sup.125 I, .sup.169 Yb and .sup.186 Re.
However, an abscess may be caused by any one of many possible pathogens, so that a radiotracer specific for a particular pathogen would have limited scope. On the other hand, infection is almost invariably accompanied by inflammation, which is a general response of the body to tissue injury. Therefore, a radiotracer specific for sites of inflammation would be expected to be useful in localizing sites of infection caused by any pathogen, as well as being useful for localizing other inflammatory sites.
One of the main phenomena associated with inflammation is the localization of leukocytes (white blood cells), usually monocytes and neutrophils, at the site of inflammation. A radiotracer specific for leukocytes would be useful in detecting leukocytes at the site of a localized infection. Currently approved nuclear medicine procedures for imaging sites of infection use either indium-111 labeled leukocytes (.sup.111 In-WBC) (see, e.g. Peters, 1992, J. Nucl. Med. 33: 65-67) or gallium-67 (.sup.67 Ga) citrate (see, e.g. Ebright et al., 1982, Arch. Int. Med. 142: 246-254). A major disadvantage of using .sup.111 In-labeled WBCs is that the preparation of the radiotracer requires a number of technical steps: sterile removal of autologous blood, sterile isolation of the leukocytes from the blood, sterile labeling of the leukocytes using conditions that do not damage the cells (since damaged WBC are taken up by the reticuloendothelial system when re-injected) and sterile return (re-injection) of the (now labeled) leukocytes to the patient. Furthermore, a delay of 12 to 48 hours between injection and imaging may be required to obtain optimum imaging. While Tc-99m labeled leukocytes have been used to shorten this delay period (see, e.g. Vorne et al., 1989, J. Nucl. Med. 30: 1332-1336), ex-corporeal labeling is still required. A preferred radiotracer would be one that either would label leukocyes in whole blood or would not require removal and manipulation of autologous blood components ex corpora.
Alternatively, .sup.67 Ga-citrate can be administered by intravenous injection. However, this compound is not specific for sites of infection or inflammation. Moreover, a delay of up to 72 hours is often required between injection of the radiotracer and imaging. In addition, the .gamma.-(gamma) emissions energies of .sup.67 Ga are not well suited to conventional gamma cameras.
Radiolabeled monoclonal and polyclonal antibodies raised against human leukocytes (including monocytes, neutrophils, granulocytes and other cell types) have been developed. Tc-99m labeled antigranulocyte monoclonal antibodies (see, e.g. Lind et al., 1990, J. Nucl. Med. 31: 417-473) and .sup.111 In-labeled non-specific human immunoglobulin (see, e.g. LaMuraglia et al., 1989, J. Vasc. Surg. 10: 20-28) have been tested for the detection of inflammation secondary to infection. .sup.111 In-labeled IgG shares the disadvantages of .sup.111 In-labeled WBC, in that 24-48 hours are required between injection and optimal imaging. In addition, radiolabeled antibodies are difficult to produce and face protracted approval procedures, as they are routinely classified as biologics by regulatory agencies.
Small, readily synthesized molecules are preferred as routinely-used radio-pharmaceuticals. There is clearly a need for small synthetic molecules that can be used either to label leukocytes in whole blood (i.e., without the need to isolate the leukocytes from the other components of whole blood) or that can be directly injected into a patient and will image sites of infection and inflammation by localizing at sites where leukocytes have accumulated.
One kind of small, readily synthesized molecule useful in such applications are peptides. The sensitivity of imaging methods using radioactively-labeled peptides is much higher than other techniques known in the art, since the specific binding of the radioactive peptide concentrates the radioactive signal over the area of interest, for example, an inflammatory site. In addition, methods for achieving high-yield chemical synthesis of small peptides are well known in the art.
Radiolabeled peptides, and in particular formyl-methionyl-leucyl-phenylalanyl (fMLF)-containing peptides, have been reported in the prior art.
Zoghbi et al., 1981, J. Nucl. Med. 22: 32 (Abst) disclose formyl peptide chemotactic factors (fMLF) derived from bacteria coupled to .sup.111 In-labeled transferrin.
Jiang et al., 1982, Nuklearmedizin 21: 110-113 disclose a chemotactic formylated peptide (fMLF) radiolabeled with .sup.125 I.
Fischman et al., 1991, J. Nucl. Med. 32: 482-491 relates to chemotactic formyl peptide (fMLF)--.sup.111 In-labeled DTPA conjugates.
EPC 90108734.6 relates to chemotactic formyl peptide (fMLF)--.sup.111 In-labeled DTPA conjugates.
U.S. Pat. No. 4,986,979 relates to the use of radiolabeled chemotactic formyl peptides (fMLF) to radiolabel leukocytes ex-corporeally via a photoaffinity label.
PCT W090/10463 relates to the use of radiolabeled chemotactic formyl peptides (fMLF) to radiolabel leukocytes ex-corporeally via a photoaffinity label.
The use of labeled fMLF peptides known in the aforementioned art suffers from the serious drawback that this peptide causes adverse physiological responses such as superoxide release from neutrophils (Niedel and Cuatrecasas, 1980, Formyl Peptide Chemotactic Receptors of Leukocytes and Macrophages, in Curr. Top. Cell. Reg. 17: 137-170), and at sufficient doses these compounds can cause leukocytopenia (Jiang et al., 1982, Nuklearmed. 21: 110-113).
Another source of inflammatory site-specific peptides is platelet factor 4, a naturally-occurring chemotactic peptide, consisting of 70 amino acids having a molecular weight of 7800 daltons and known in the prior art to bind to neutrophils and monocytes, cell types known to be associated with sites of inflammation and infection in vivo. Of interest with regard to the present invention, the carboxyl terminus of PF4 is known to bind to polysulfated glycans such as heparin (Loscalzo et al., 1985, Arch. Biochem. Biophys. 240: 446-455). In addition, an advantage of PF4 over the fMLF compounds known in the art is that it does not cause superoxide release from neutrophils even at concentrations of 20 .mu.M (Bebawy et al., 1986, J. Leukocyte Biol. 39: 423-434).
Thorbecke & Zucker, 1989, European Patent Application No. 88111962.2 disclose compositions and methods for modulating immune responses comprising administering an immunomodulating amount of platelet factor 4 or peptides derived therefrom.
Deuel et al., 1977, Proc. Natl. Acad. Sci. USA 74: 2256-2258 disclose the amino acid sequence of human platelet factor 4.
Deuel et al., 1981, Proc. Natl. Acad. Sci. USA 78: 4584-4587 disclose that platelet factor 4 is chemotactic for neutrophils and monocytes in vitro.
Osterman et al., 1982, Biochem. Biophys. Res. Comm. 107: 130-135 disclose that the carboxyl-terminal tridecapeptide of platelet factor 4 has chemotactic properties.
Holt & Niewiarowski, 1985, Sem. Hematol. 22: 151-163 provide a review of the biochemistry of platelet .alpha.-granule proteins, including platelet factor 4.
Goldman et al., 1985, Immunol. 54: 163-171 reveal that fMLF receptor-mediated uptake is inhibited in human neutrophils by platelet factor 4 and a carboxyl-terminal dodecapeptide thereof.
Loscalzo et al., ibid., describe the biochemical interaction between platelet factor 4 and glycosaminoglycans such as heparin.
Bebawy et al., ibid., describe the platelet factor 4-mediated chemotactic response of neutrophils in vitro.
Maione et al., 1989, Science 247: 77-79 disclose that angiogenesis is inhibited by recombinant human platelet factor 4 and peptide fragments thereof.
The use of chelating agents for radiolabeling polypeptides, and methods for labeling peptides and polypeptides with Tc-99m are known in the prior art and are disclosed in co-pending U.S. patent application Ser. No. 07/653,012, now abandoned, which issued as U.S. Pat. No. 5,654,272; Ser. No. 07/807,062, now U.S. Pat. No. 5,443,815; Ser. No. 07/871,282, a divisional of which issued as U.S. Pat. No. 5,720,934; Ser. No. 07/886,752, now abandoned, a continuation of which has been allowed as U.S. Ser. Nos. 08/273,274; 07/893,981, now U.S. Pat. No. 5,508,020; Ser. No. 07/955,466; Ser. No. 08/019,864, now U.S. Pat. No. 5,552,525; Ser. No. 08/044,825, now abandoned, which issued as U.S. Pat. No. 5,645,815; and Ser. No. 08/073,577, now U.S. Pat. No. 5,561,220; and PCT International Applications PCT/US92/00757, PCT/US92/10716, PCT/US93/02320 and PCT/US93/04794, which are hereby incorporated by reference.